Bottom Line:
Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM.The study also showed that AMP is another possible exchange substrate.The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

Affiliation: Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.

ABSTRACTIn cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, HvBT1. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

pone-0098524-g007: Transport activity of HvBT1 in intact E. coli cells.Escherichia coli C43 cells harboring the recombinant plasmid and the empty one as a control were incubated with different concentrations of [α-32P] ADP-Glc. The cells were incubated at 30°C for 10 min. The control values have been subtracted. The data are the mean ± SE of three independent experiments, each with three replicates. A: Km value of ADP-glucose is 614.5±33.24 µM and Vmax of 254.14 ±19.45 nmol of ADP-Glc mg of protein−1 h−1. B: Km and Vmax values of ADP is 334.7±39.3 µM and of 47.07±3.51 nmol of ADP-Glc mg of protein−1 h−1, respectively.

Mentions:
The transport of [α-32P] ADP-Glc was studied using intact E. coli cells harboring the expression plasmid containing HvBT1 or the control plasmid (with no HvBT1). The results showed that the import of the α-32P labeled substrate follows a non-linear regression trend for Michaelis-Menten kinetics (Figure 7). The affinity of HvBT1 for ADP- glucose was analyzed with different concentrations of [α-32P] ADP-glucose using Wolfram Mathematica 8.0 software (Wolfram, Champaign, IL, USA). Increasing the substrate concentration led to increased radiolabeled ADP-glucose uptake into the intact E. coli cells expressing the HvBT1. The Km value of ADP-Glc was calculated to be 614.5 µM and the Vmax to be 254.1 nmol of ADP-Glc mg of protein−1 h−1 (Figure 7A). Uptake for [α-32P] ADP was also analyzed as described for [α-32P] ADP-Glc. Likewise; an increase in the import rate of [α-32P] ADP was observed with increased concentrations of [α-32P] ADP. The Km and Vmax values for ADP are 334.7 µM and 74.07 nmol of ADP mg protein−1 h−1, respectively (Figure 7B).

pone-0098524-g007: Transport activity of HvBT1 in intact E. coli cells.Escherichia coli C43 cells harboring the recombinant plasmid and the empty one as a control were incubated with different concentrations of [α-32P] ADP-Glc. The cells were incubated at 30°C for 10 min. The control values have been subtracted. The data are the mean ± SE of three independent experiments, each with three replicates. A: Km value of ADP-glucose is 614.5±33.24 µM and Vmax of 254.14 ±19.45 nmol of ADP-Glc mg of protein−1 h−1. B: Km and Vmax values of ADP is 334.7±39.3 µM and of 47.07±3.51 nmol of ADP-Glc mg of protein−1 h−1, respectively.

Mentions:
The transport of [α-32P] ADP-Glc was studied using intact E. coli cells harboring the expression plasmid containing HvBT1 or the control plasmid (with no HvBT1). The results showed that the import of the α-32P labeled substrate follows a non-linear regression trend for Michaelis-Menten kinetics (Figure 7). The affinity of HvBT1 for ADP- glucose was analyzed with different concentrations of [α-32P] ADP-glucose using Wolfram Mathematica 8.0 software (Wolfram, Champaign, IL, USA). Increasing the substrate concentration led to increased radiolabeled ADP-glucose uptake into the intact E. coli cells expressing the HvBT1. The Km value of ADP-Glc was calculated to be 614.5 µM and the Vmax to be 254.1 nmol of ADP-Glc mg of protein−1 h−1 (Figure 7A). Uptake for [α-32P] ADP was also analyzed as described for [α-32P] ADP-Glc. Likewise; an increase in the import rate of [α-32P] ADP was observed with increased concentrations of [α-32P] ADP. The Km and Vmax values for ADP are 334.7 µM and 74.07 nmol of ADP mg protein−1 h−1, respectively (Figure 7B).

Bottom Line:
Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM.The study also showed that AMP is another possible exchange substrate.The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

Affiliation:
Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.

ABSTRACTIn cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, HvBT1. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.